Automated DNA sequencing technique

ABSTRACT

A system for the electrophoretic analysis of DNA fragments produced in DNA sequencing operations comprising: a source of chromophore or fluorescent tagged DNA fragments; a zone for contacting an electrophoresis gel; means for introducing said tagged DNA fragments to said zone; and photometric means for monitoring said tagged DNA fragments as they move through said gel.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of Ser. No. 106,232, filed Oct. 7,1987, abandoned, which in turn was a continuation-in-part of U.S. Ser.No. 722,742, filed Apr. 11, 1985 (now abandoned) which was acontinuation-in-part of U.S. Ser. No. 689,013 filed Jan. 2, 1985 (nowabandoned) which, in turn, was a continuation-in-part of U.S. Ser. No.570,973 filed Jan. 16, 1984, now abandoned.

BACKGROUND OF THE INVENTION

The development of reliable methods for sequence analysis of DNA(deoxyribonucleic acid) and RNA (ribonucleic acid) has been one of thekeys to the success of recombinant DNA and genetic engineering. Whenused with the other techniques of modern molecular biology, nucleic acidsequencing allows dissection and analysis of animal, plant and viralgenomes into discrete genes with defined chemical structure. Since thefunction of a biological molecule is determined by its structure,defining the structure of a gene is crucial to the eventual manipulationof this basic unit of hereditary information in useful ways. Once genescan be isolated and characterized, they can be modified to producedesired changes in their structure that allow the production of geneproducts--proteins--with different properties than those possessed bythe original proteins. Microorganisms into which the natural orsynthetic genes are placed can be used as chemical "factories" toproduce large amounts of scarce human proteins such as interferon,growth hormone, and insulin. Plants can be given the genetic informationto allow them to survive harsh environmental conditions or produce theirown fertilizer.

The development of modern nucleic acid sequencing methods involvedparallel developments in a variety of techniques. One was the emergenceof simple and reliable methods for cloning small to medium-sized strandsof DNA into bacterial plasmids, bacteriophages, and small animalviruses. This allowed the production of pure DNA in sufficientquantities to allow its chemical analysis. Another was the nearperfection of gel electrophoretic methods for high resolution separationof oligonucleotides on the basis of their size. The key conceptualdevelopment, however, was the introduction of methods of generatingsize-nested sets of fragments cloned, purified DNA that contain, intheir collection of lengths, the information necessary to define thesequence of the nucleotides comprising the parent DNA molecules.

Two DNA sequencing methods are in widespread use. These are the methodof Sanger, F., Nicken, S. and Coulson, A. R. Proc. Natl. Acad. Sci. USA74, 5463 (1977) and the method of Maxam, A. M. and Gilbert, W. Methodsin Enzymology 65, 499-599 (1980).

The method developed by Sanger is referred to as the dideoxy chaintermination method. In the most commonly used variation of this method,a DNA segment is cloned into a single-stranded DNA phage such as M13.These phage DNAs can serve as templates for the primed synthesis of thecomplementary strand by the Klenow fragment of DNA polymerase I. Theprimer is either a synthetic oligonucleotide or a restriction fragmentisolated from the parental recombinant DNA that hybridizes specificallyto a region of the M13 vector near the 3" end of the cloned insert. Ineach of four sequencing reactions, the primed synthesis is carried outin the presence of enough of the dideoxy analog of one of the fourpossible deoxynucleotides so that the growing chains are randomlyterminated by the incorporation of these "deadend" nucleotides. Therelative concentration of dideoxy to deoxy forms is adjusted to give aspread of termination events corresponding to all the possible chainlengths that can be resolved by gel electrophoresis. The products fromeach of the four primed synthesis reactions are then separated onindividuals tracks of polyacrylamide gels by the electrophoresis.Radioactive tags incorporated in the growing chains are used to developan autoradiogram image of the pattern of the DNA in each electrophoresistrack. The sequence of the deoxynucleotides in the cloned DNA isdetermined from an examination of the pattern of bands in the fourlanes.

The method developed by Maxam and Gilbert uses chemical treatment ofpurified DNA to generate size-nested sets of DNA fragments analogous tothose produced by the Sanger method. Single or double-stranded DNA,labeled with radioactive phosphate at either the 3' or 5' end, can besequenced by this procedure. In four sets of reactions, cleavage isinduced at one or two of the four nucleotide bases by chemicaltreatment. Cleavage involves a threestage process: modification of thebase, removal of the modified base from its sugar, and strand scissionat that sugar. Reaction conditions are adjusted so that the majority ofend-labeled fragments generated are in the size range (typically 1 to400 nucleotides) that can be resolved by gel electrophoresis. Theelectrophoresis, autoradiography, and pattern analysis are carried outessentially as is done for the Sanger method. (Although the chemicalfragmentation necessarily generates two pieces of DNA each time itoccurs, only the piece containing the end label is detected on theautoradiogram.)

Both of these DNA sequencing methods are in widespread use, and each hasseveral variations.

For each, the length of sequence that can be obtained from a single setof reactions is limited primarily by the resolution of thepolyacrylamide gels used for electrophoresis. Typically, 200 to 400bases can be read from a single set of gel tracks. Although successful,both methods have serious drawbacks, problems associated primarily withthe electrophoresis procedure. One problem is the requirement of the useof radiolabel as a tag for the location of the DNA bands in the gels.One has to contend with the short half-life of phosphorus-32, and hencethe instability of the radiolabeling reagents, and with the problems ofradioactive disposal and handling. More importantly, the nature ofautoradiography (the film image of a radioactive gel band is broaderthan the band itself) and the comparison of band positions between fourdifferent gel tracks (which may or may not behave uniformly in terms ofband mobilities) can limit the observed resolution of bands and hencethe length of sequence that can be read from the gels. In addition, thetrack-to-track irregularities make automated scanning of theautoradiograms difficult--the human eye can presently compensate forthese irregularities much better than computers can. This need formanual "reading" of the autoradiograms is time-consuming, tedious anderror-prone. Moreover, one cannot read the gel patterns while theelectrophoresis is actually being performed, so as to be able toterminate the electrophoresis once resolution becomes insufficient toseparate adjoining bands, but must terminate the electrophoresis at somestandardized time and wait for the autoradiogram to be developed beforethe sequence reading can begin.

The invention of the present patent application addresses these andother problems associated with DNA sequencing procedures and is believedto represent a significant advance in the art. The preferred embodimentof the present invention represents a further and distinct improvement.

SUMMARY OF THE INVENTION

Briefly, this invention comprises a novel process for theelectrophoretic analysis of DNA fragments produced in DNA sequencingoperations wherein chromophores or fluorophores are used to tag the DNAfragments produced by the sequencing chemistry and permit the detectionand characterization of the fragments as they are resolved byelectrophoresis through a gel. The detection employs an absorption orfluorescent photometer capable of monitoring the tagged bands as theyare moving through the gel.

This invention further comprises a novel process for the electrophoreticanalysis of DNA fragments produced in DNA sequencing operations whereina set of four chromophores are used to tag the DNA fragments produced bythe sequencing chemistry and permit the detection and characterizationof the fragments as they are resolved by electrophoresis through a gel;the improvement wherein the four different fragment sets are tagged withthe fluorophores fluorescein, Texas Red, tetramethyl rhodamine, and7-nitro-benzofurazan.

This invention also includes a novel system for the electrophoreticanalysis of DNA fragments produced in DNA sequencing operationscomprising:

a source of chromophore or fluorescent tagged DNA fragments.

a zone for containing an electrophoresis gel,

means for introducing said tagged DNA fragments to said zone; and

photometric means for monitoring or detecting said tagged DNA fragmentsas they move through and are separated by said gel.

It is an object of this invention to provide a novel process for thesequence analysis of DNA.

It is another object of our invention to provide a novel system for theanalysis of DNA fragments.

More particularly, it is an object of this invention to provide animproved process for the sequence analysis of DNA.

These and other objects and advantages of this invention will beapparent from the detailed description which follows.

DETAILED DESCRIPTION OF THE INVENTION

Turning to the drawings:

FIG. 1 is an illustration of one means of end-labeling a DNA fragmentwith a fluorescent tag. Pst. I and T4 DNA ligase are enzymes commonlyused in recombinant DNA research.

FIG. 1A pertains to PST I DNA ligase. FIG. 1B pertains to T4 DNA ligase.

FIG. 2 is a block diagram of automated DNA sequencer, gelelectrophoretic system.

FIG. 3 is a comparison of the type of data produced by DNA sequencing ofthe sequence shown in FIG. 1.

FIG. 3I shows a hypothetical DNA sequence.

FIG. 3II shows an idealized autoradiogram of polyacrylamide slab gelproduced by the chain termination method according to the prior art.

FIG. 3III shows an idealized diagram of DNA on an acrylamide gel,produced according to the present invention.

FIG. 3IV shows an idealized output from the detection on colored bandson the acrylamide gel of FIG. 3III.

FIG. 4 is a block diagram of a preferred DNA sequencer according to thisinvention.

FIG. 5 shows the emission spectra for the four fluorophores used as tagsin the preferred embodiment of this invention.

FIG. 6 is a schematic diagram of a possible optical configuration in thedetector unit. P, lamp source; L1, objective lens; L2, collimating lens;F1, UV blocking filter; F2, heat blocking filter; F3, band passexcitation filter; F4, long pass emission filter; DM, dichroic mirror;C, polyacrylamide gel; PMT, photomultiplier tube.

FIG. 7 is a schematic diagram of another possible optical configurationin the detector unit. F1 to F4 are bandpass filters centered at theemission maximum of the different dyes. P1 to P4 are photomultipliertubes. The excitation light is of a wavelength such that it is nottransmitted through any of the filters F1 to F4.

In the previous methods of DNA sequencing, including those based on theSanger dideoxy chain termination method, a single radioactive label,phosphorus-32, is used to identify all bands on the gels. Thisnecessitates that the fragment sets produced in the four synthesisreactions be run on separate gel tracks and leads to the problemsassociated with comparing band mobilities in the different tracks. Thisproblem is overcome in the present invention by the use of a set of fourchromophores or fluorophores with different absorption or fluorescentmaxima, respectively. Each of these tags is coupled chemically to theprimer used to initiate the synthesis of the fragment strands. In turn,each tagged primer is then paired with one of the dideoxynucleotides andused in the primed synthesis reaction with the Klenow fragment of DNApolymerase.

The primers must have the following characteristics. 1) They must have afree 3'hydroxyl group to allow chain extension by the polymerase. 2)They must be complementary to a unique region 3' of the cloned insert.3) They must be sufficiently long to hybridize to form a unique, stableduplex. 4) The chromophore or fluorophore must not interfere with thehybridization or prevent 3'-end extension by the polymerase.

Conditions 1, 2 and 3 above are satisfied by several syntheticoligonucleotide primers which are in general use for Sanger-typesequencing utilizing M13 vectors.

One such primer is the 15 mer 5' CCC AG TCA CGA CGT T 3' where A, C, Gand T represent the four different nucleoside components of DNA; A,adenosine; C, cytosine; G, guanosine; T, thymidine.

In the preferred embodiment of the present invention a set of fourfluorophores with different emission spectra, respectively, are used.These different emission spectra are shown in FIG. 5. Each of these tagsis coupled chemically to the primer used to initiate the synthesis ofthe fragment strands. In turn, each tagged primer is then paired withone of the dideoxynucleotides and used in the primed synthesis reactionwith the Klenow fragment of DNA polymerase.

The dyes used must have high extinction coefficients and/or reasonablyhigh quantum yields for fluorescence. They must have well resolvedadsorption maxima and/or emission masima. Representative of such aminoreactive dues are: fluorescein isothiocyanage (FITC, λ_(max) ^(Ex) =495,λ_(max) ^(Em) =520, ε₄₉₅ ≃8×10⁴), tetramethyl rhodamine isothiocyanate(TMRITC, λ_(max) ^(Ex) =550, λ_(max) ^(Em) =578, ε₅₅₀ ≃4×10⁴), andsubstituted rhodamine isothiocyanate (XRITC, λ=580, λ_(max) ^(Em) =604,ε₅₈₀ ≃8×10⁴) where λ represents the wavelength in nanometers, Ex isexcitation, Em is emission, max is maximum, and ε is the molarextinction coefficient. These dyes have been attached to the M13 primerand the conjugates electrophoresed on a 20% polyacrylamide gel. Thelabeled primers are visible by both their absorption and theirfluorescence in the gel. All four labeled primers have identicalelectrophoretic mobilities. The dye conjugated primers retain theirability to specifically hybridize to DNA, as demonstrated by theirability to replace the underivitized oligonucleotide normally used inthe sequencing reactions.

The chemistry for the coupling of the chromophoric or fluorophoric tagsis described in assignee's copending patent applications Ser. No.565,010, filed Dec. 20, 1983, now abandoned, and Ser. No. 709,579, filedMar. 8, 1985, (now abandoned), and Ser. No. 878,045 filed Jun. 24, 1985,and now issued as U.S. Pat. No. 4,849,513 the disclosures of which areexpressly incorporated herein by reference. The strategy used is tointroduce an aliphatic amino group at the 5' terminus as the lastaddition in the synthesis of the oligonucleotide primer. This reactiveamino group may then readily be coupled with a wide variety of aminoreactive fluorophores or chromophores. This approach aids compatibilityof the labeled primers with condition 4 above.

End labeling of DNA for use with Maxam/Gilbert method. In theMaxam/Gilbert method of DNA sequencing, the end of the piece of DNAwhose sequence is to be determined must be labeled. This isconventionally done enzymatically using radioactive nucleosides. Inorder to use the Maxam/Gilbert method in conjunction with the dyedetection scheme described in this invention, the DNA piece must belabeled with dyes. One manner in which this may be accomplished is shownin FIG. 1. Certain restriction endonucleases generate what is known as a3' overhang as the product of DNA cleavage. These enzymes generate a"sticky end," a short stretch of single stranded DNA at the end of apiece of double stranded DNA. This region will anneal with acomplementary stretch of DNA, which may be covalently joined to theduplex DNA with the enzyme ligase. In this manner one of the strands iscovalently linked to a detectable moiety. This moiety may be a dye, anamino group or a protected amino group (which could be deprotected andreacted with dye subsequent to the chemical reactions).

Sequencing reactions. The dideoxy sequencing reactions are performed inthe standard fashion Smith, A. J. H., Methods in Enzymology 65, 56-580(1980), except that the scale may be increased if necessary to providean adequate signal intensity in each band for detection. The reactionsare done using a different color primer for each different reaction. Noradiolabeled nucleoside triphosphate need be included in the sequencingreaction.

The Maxam/Gilbert sequencing reactions are performed in the usualmanner, Gil, S.F.Aldrichimica Acta 16(3), 59-61 (1983), except that theend label is either one or four colored dyes, or a free or protectedamino group which may be reacted with dye subsequently.

Detection. There are many different ways in which the tagged moleculeswhich have been separated by length using polyacrylamide gelelectrophoresis may be detected. Four illustrative modes are describedbelow. These are i) detection of the fluorescence excited by light ofdifferent wavelengths for the different dyes, ii) detection offluorescence excited by light of the same wavelength for the differentdyes, iii) elution of the molecules from the gel and detection bychemiluminescence, and iv) detection by the absorption of light bymolecules. In modes i) and ii) the fluorescence detector should fulfillthe following requirements. a) The excitation light beam should not havea height substantially greater than the height of a band. This isnormally in the range of 0.1 to 0.5 mm. The use of such a narrowexcitation beam allows the attainment of maximum resolution of bands. b)The excitation wavelength can be varied to match the absorption maximaof each of the different dyes or can be a single narrow, high intensitylight band that excites all four fluorophores and does not overlap withany of the fluorescence emission. c) The optical configuration shouldminimize the flux of scattered and reflected excitation light to thephotodetector 14. The optical filters to block out scattered andreflected excitation light are varied as the excitation wavelength isvaried. d) The photodetector 14 should have a fairly low noise level anda good spectral response and quantum efficiency throughout the range ofthe emission of the dyes (500 to 600 nm for the dyes listed above). e)The optical system for collection of the emitted fluorescence shouldhave a high numerical aperture. This maximizes the fluorescence signal.Furthermore, the depth of field of the collection optics should includethe entire width of the column matrix.

Two illustrative fluorescence detection systems are diagrammed in FIGS.6 and 7. The system in FIG. 6 is compatible with either singlewavelength excitation or multi wavelength excitation. For singlewavelength excitation, the filter F4 is one of four band pass filterscentered at the peak emission wavelength of each of the dyes. Thisfilter is switched every few seconds to allow continual monitoring ofeach of the four fluorophores. For multi wavelength excitation, theoptical elements F3 (excitation filter), DM (dichroic mirror), and F4(barrier filter) are switched together. In this manner both theexcitation light and the observed emission light are varied. The systemin FIG. 7 is a good arrangement for the case of single wavelengthexcitation. This system has the advantage that no moving parts arerequired, and fluorescence from all four of the dyes may besimultaneously and continuously monitored. A third approach (iii above)to detection is to elute the labeled molecules at the bottom of the gel,combine them with an agent for excitation of chemiluminescence such as1,2 dioxetane dione, Gill, S.K. Aldrichimica Acta 16(3), 59-61 (1983);Mellbin, G. J. Liq. Chrom. 6(9), 1603-1616 (1983), and flow the mixturedirectly into a detector which can measure the emitted light at fourseparate wavelengths. The background signal in chemiluminescence is muchlower than in fluorescence, resulting in higher signal to noise ratiosand increased sensitivity. Finally, the measurement may be made bymeasurements of light absorption (iv above). In this case, a light beamof variable wavelength is passed through the gel, and the decrease inthe beam intensity due to absorption of light at the differentwavelengths corresponding to the absorption maximum of the four dyes, itis possible to determine which dye molecule is in the light path. Asdisadvantage of this type of measurement is that absorption measurementsare inherently less sensitive than fluorescence measurements.

The above-described detection system is interfaced to a computer 16. Ineach time interval examined, the computer 16 receives a signalproportional to the measured signal intensity at that time for each ofthe four colored tags. This information tells which nucleotideterminates the DNA fragment of the particular length in the observationwindow at that time. The temporal sequence of colored bands gives theDNA sequence. In FIG. 3 is shown the type of data obtained byconventional methods, as well as the type of data obtained by theimprovements described in this invention.

The following Examples are presented solely to illustrate the invention.In the Examples, parts and percentages are by weight unless otherwiseindicated.

EXAMPLE I

Gel electrophoresis. Aliquots of the sequencing reactions are combinedand loaded onto a 5% polyacrylamide column 10 shown in FIG. 2 from theupper reservoir 12. The relative amounts of the four different reactionsin the mixture are empirically adjusted to give approximately the samefluorescence or absorptive signal intensity from each of the dye DNAconjugates. This permits compensation for differences in dye extinctioncoefficients, dye fluorescence quantum yields, detector sensitivitiesand so on. A high voltage is placed across the column 10 so as toelectrophorese the labeled DNA fragments through the gel. The labeledDNA segments differing in length by a single nucleotide are separated byelectrophoresis in this gel matrix. At or near the bottom of the gelcolumn 10, the bands of DNA are resolved from one another and passthrough the detector 14 (more fully described above). The detector 14detects the fluorescent or chromophoric bands of DNA in the gel anddetermines their color, and therefore to which nucleotide theycorrespond. This information yields the DNA sequence.

EXAMPLE II

FIG. 4 shows a block diagram of a DNA sequenator for use with one dye ata time. The beam (4880 A) from an argon ion laser 100 is passed into thepolyacrylamide gel tube (sample) 102 by means of a beamsteerer 104.Fluorescence exited by the beam is collected using a low f-number lens106, passed through an appropriate set of optical filters 108 and 110 toeliminate scattered excitation light and detected using aphotomultiplier tube (PMT) 112. The signal is readily detected on astrip chart recorder. DNA sequencing reactions are carried out utlizinga fluorescein labeled oligonucletide primer. The peaks on the chartcorrespond to fragments to fluorescein labeled DNA of varying lengthssynthesized in the sequencing reactions and separated in the gel tube byelectrophoresis. Each peak contains on the order of 10⁻¹⁵ to 10⁻¹⁶ molesof fluorescein, which is approximately equal to the amount of DNAobtained per band in an equivalent sequencing gel utilizing radioisotopedetection. This proves that the fluorescent tag is not removed ordegraded from the oligonucleotide primer in the sequencing reactions. Italso demonstrates that the detection sensitivity is quite adequate toperform DNA sequence analysis by this means.

MATERIALS

Fluorescein-5-isothiocyanate (FITC) and Texas Red were obtained fromMolecular Probes, Inc. (Junction City, Oreg.). Tetramethyl rhodamineisothiocyanate (TMRITC) was obtained from Research Organics, Inc.(Cleveland, Ohio). 4-fluoro-7-nitro-benzofurazan (NBD-fluoride) wasobtained from Sigma Chemical Co. (St. Louis, Mo.). Absorption spectrawere obtained on a H/P 8491 spectrophotometer. High performance liquidchromatography was performed on a system composed of two Altex 110Apumps, a dual chamber gradient mixer, Rheodyne injector, Kratos 757 UVdetector, and an Axxiom 710 controller.

EXAMPLE III Addition of 5'-aminothymidine phosphoramidites tooligonuleotides

The protected 5'-aminothymidine phosphoramidites,5'-(N-9-fluorenylmethyloxycarbonyl)-5'-amino-5'-deoxy-3'-N,N-diisopropylaminomethoxyphosphinylthymidine, is coupled to the 5'-hydroxyl of an oligonucleotide usingwell established DNA synthetic procedures. The solvents and reactionconditions used are identical to those used in oligonucleotidesynthesis.

EXAMPLE IV Dye conjugation

The basic procedure used for the attachment of fluorescent dye moleculesto the amino oligonucleotides is to combine the amino oligonucleotideand the dye in aqueous solution buffered to pH 9, to allow the reactionto stand at room temperature for several hours, and then to purify theproduct in two stages. The first purification step is to remove the bulkof the unreacted or hydrolyzed dye by gel filtration. The secondpurification stage is to separate the dye conjugate from unreactedoligonucleotide by reverse phase high performance liquid chromatography.Slight variations upon these conditions are employed for the differentdyes, and the specific procedures and conditions used for fourparticular dyes are given below and in Table 1.

                  TABLE 1                                                         ______________________________________                                        Reverse Phase HPLC Conditions for                                             Dye-oligonucleotide Purification                                              Sample          Retention time                                                ______________________________________                                        PLP-15.sup.a    18'                                                           PLP-15-T-NH.sub.2 .sup.b                                                                      18'                                                           FITC PLP-15.sup.c                                                                             27'                                                           NBD PLP-15      25'                                                           TMRITC PLP-15   32' and 34'.sup.d                                             Texas Red PLP-15                                                                              42'                                                           ______________________________________                                         .sup.a PLP-15 is an oligonucleotide primer for DNA sequence analysis in       the M13 vectors. Its sequence is 5'CCC AGT CAC GAC FTT 3'.                    .sup.b PLP-15-T-NH.sub.2 is the oligonucleotide PLP15 to which a              5'-amino5'-deoxythymidine base has been added to==at the 5' terminus.         .sup.c The nomenclature Dye PLP15 signifies the conjugate of                  PLP15-T-NH.sub.2 and the dye molecule.                                        .sup.d Two fluorescent oligonucleotide products were obtained with TMRITC     Both were equally effective in sequencing. This is presumed to be due to      the two isomers of TMRITC which are present in the commercially available     material.                                                                

Retention times shown are for HPLC gradients of 20% solvent B/80%solvent A to 60% solvent B/40% solvent A in 40 min., where solvent A is0.1M triethylammonium acetate pH 7.0 and solvent B is 50% acetonitrile,50% 0.1M triethylammonium acetate pH 7.0. The column was an Axxiom ODS 5micron C 18 column #555-102 available from Cole Scientific, Calabasas,Calif. This gradient is not optimized for purification of PLP-15 andPLP-15-T-NH₂, but the retention times are included for comparison withthe dye primer conjugates.

The following procedure is for use with fluorescein isothiocyanate or4-fluoro-7-nitro-benzofurazan. Amino oligonucleotide (0.1 ml of ˜1 mg/mloligonucleotide in water) is combined with 1M sodiumcarbonate/bicarbonate buffer pH 9 (50 μl), 10 mg/ml dye indimethylformamide (20 μl) and H₂ O (80 μl). This mixture is kept in thedark at room temperature for several hours. The mixture is applied to a10 ml column of Sephadex G-25 (medium) and the colored band of materialeluting in the excluded volume is collected. The column is equilibratedand run in water. In control reactions with underivatizedoligonucleotides, very little if any dye is associated with theoligonucleotide eluting in the void volume. The colored material isfurther purified by reverse phase high performance liquid chromatographyon an Axxiom C₁₈ column (#555-102, Cole Scientific, Calabasas, Calif.)in a linear gradient of acetonitrile:0.1M triethylammonium acetate, pH7.0. It is convenient for this separation to run the column eluantthrough both a UV detector (for detecting the DNA absorbance) and afluorescence detector (for detecting the dye moiety). The desiredproduct is a peak on the chromatogram which is both strongly UVabsorbing and strongly fluorescent. The dye oligonucleotide conjugateselute at higher acetonitrile concentrations than the oligonucleotidesalone, as shown in Table 1. The oligonucleotide is obtained from thehigh performance liquid chromatographyin solution in a mixture ofacetonitrile and 0.1M triethylammonium acetate buffer. This is removedby lyophilization and the resulting material is redissolved by vortexingin 10 mM sodium hydroxide (for a minimum amount of time) followed byneutralization with a five fold molar excess (to sodium hydroxide) ofTris buffer, pH 7.5.

The conjugation with Texas Red is identical to that described forfluorescein isothiocyanate and 4-fluoro-7-nitro-benzofurazan, exceptthat:

a) prior to separation on Sephadex G-25 the reaction is made 1M inammonium acetate and kept at room temperature for 30 minutes, and

b) the Sephadex G-25 column is run in 0.1M ammonium acetate. Thislargely eliminates nonspecific binding of the dye molecule to theoligonucleotide.

The conjugation with tetramethyl rhodamine isothiocyanate is identicalto that for Texas Red except that the reaction is carried out in 10 mMsodium carbonate/bicarbonate buffer, pH 9.0, and 50% dioxane. Thisincreases solubility of the tetramethyl rhodamine and a much higheryield of dye oligonucleotide conjugate is obtained.

In some cases, particularly with the rhodamine-like dyes, a substantialamount of nonspecific binding of dye was observed, as manifested by aninappropriately large dye absorption present in the material eluted fromthe gel filtration column. In these cases the material was concentratedand reapplied to a second gel filtration column prior to highperformance liquid chromatography purification. This generally removedthe majority of the noncovalently associated dye.

EXAMPLE V Properties of dye-oligonucleotide conjugates

The development of chemistry for the synthesis of dye oligonucleotideconjugates allows their use as primers in DNA sequence analysis. Variousfluorescent dye primers have been tested by substituting them for thenormal primer in DNA sequence analysis by the enzymatic method. Anautoradiogram of a DNA sequencing gel in which these dye-conjugatedprimers were utilized in T reactions in place of the normaloligonucleotide primer was prepared. This autoradiogram was obtained byconventional methods employing α- ³² PdCTP as a radiolabel. Theautoradiogram showed that the underivitized primer, amino-derivitizedprimer, and dye conjugated primers all give the same pattern of bands(corresponding to the DNA sequence), indicating that the derivitizedprimers retain their ability to hybridize specifically to thecomplementary strand. Secondly, the bands generated using the differentprimers differ in their mobilities, showing that it is indeed thedye-primers which are responsible for the observed pattern, and not acontaminant of unreacted or underivitized oligonucleotide. Thirdly, theintensity of the bands obtained with the different primers iscomparable, indicating that the strength of hybridization is notsignificantly perturbed by the presence of the dye molecules.

The separations are again carried out in an acrylamide gel column. Thebeam from an argon ion laser is passed into the polyacrylamide gel tube(sample) by means of a beamsteerer. Fluorescence exited by the beam iscollected using a low f-number lens, passed through an appropriate setof optical filters to eliminate scattered excitation light and detectedusing a photomultiplier tube (PMT). The signal is monitored on a stripchart recorder. DNA sequencing reactions have been carried out utilizingeach of the four different dye coupled oligonucleotide primers. In eachcase a series of peaks are observed on the chart paper. The peakscorrespond to fragments of dye labeled DNA of varying lengthssynthesized in the sequencing reactions and separated in the gel tube byelectrophoresis. Each peak contains of the order of 10⁻¹⁴ to 10⁻¹⁶ molesof dye, which is approximately equal to the amount of DNA obtained perband in an equivalent sequencing gel utilizing radioisotope detection.This proves that the fluorescent tag is not removed or degraded from theoligonucleotide primer in the sequencing reactions. It also demonstratesthat the detection sensitivity is quite adequate to perform DNA sequenceanalysis by this means, and that adequate resolution of the DNAfragments is obtained in a tube gel system.

Having fully described the invention it is intended that it be limitedonly by the lawful scope of the appended claims.

We claim:
 1. A novel system for the electrophoretic analysis ofoligonucleoide fragments produced in sequencing operations comprising:asource of chromophore of fluorescent tagged oligonucleotide fragments,said chromophore or fluorophore being distinguishable by its spectralcharacteristics, a zone for containing an electrophoresis medium, meansfor introducing said tagged oligonucleotide fragments to said zone; andphotometric means for monitoring and distinguishing said tagged DNAfragments upon separation in said medium.
 2. The novel system of claim 1wherein the photometric means is an absorption photometer.
 3. The novelsystem of claim 1 wherein the photometric means is a fluorescentphotometer.
 4. The novel system of claim 1 wherein the oligonucleotidefragments are labeled with an amino group which is coupled to a dyemolecule.
 5. The novel system of claim 1 wherein a set of fourchromophores or fluorophores are present to tag said oligonucleotide. 6.The novel system of claim 1 wherein said source of taggedoligonucleotide fragments is positioned at one end of said zone, andsaid detector is positioned in proximity to the opposite end of saidzone.
 7. A novel system for the electrophoretic analysis of DNAfragments produced in DNA sequencing operations by the chain terminationmethod, comprising:a source of chromophore or fluorescent tagged DNAfragments from sequencing operations wherein the primer oligonucleotidefrom each of the four sequencing reactions A, C, G, and T has adifferent chromophore or fluorescent tag attached to it, each tag beingdistinguishable from the others by its spectral characteristics. a zonecontaining an electrophoresis medium; means for introducing said taggedDNA fragments to said zone; and photometric means for monitoring anddistinguishing said tagged DNA fragments upon separation in said medium.